Abstract Through this project we propose to develop a therapeutic antibody against Staphylococcus aureus (S.a.) that simultaneously targets multiple secreted S.a. toxins, as well as a cell-surface target, in a molecule that also contains a functional human IgG1 Fc domain. S. aureus infections are very common and their treatment is becoming more complex due to broadening antibiotic resistance. Inhibrx has developed a platform where multiple camelid heavy chain only variable domains (VHHs) are joined end to end, together with an Fc effector domain, in any order to produce multi-valent, multi-specific molecules targeting multiple epitopes on a single target or multiple targets. We have already developed a tetravalent, bispecific heavy chain only antibody, with specificity against five S.a. toxins. We propose to combine this antibody with a bacterial surface binding component to create a multi-specific anti-?MRSA antibody. We hypothesize that toxin blocking functionality will protect immune cells recruited to the site of infection, while cell binding capability will enable opsonization of S.a., followed by its clearance.? We have targeted two highly conserved S.a. cell surface proteins for development of antibodies: Protein A and ?EFB, and identified a number of candidate high affinity antibodies against either of these targets. Importantly, as Protein A binds the Fc region of antibodies in a non-immune fashion, preventing efficient opsonization of the bacterium, Inhibrx has integrated a proprietary Fc domain that does not bind Protein A into our anti-Protein A targeting antibodies, producing antibodies that avoid non- immune Protein A binding. In the present application we propose to select bacterial binding antibodies from among the above candidates, and use them to create hexavalent, multi-specific antibodies that incorporate toxin blocking VHHs targeting five S.a. toxins, together with cell surface-binding VHHs, and a Protein A resistant Fc domain, all in one antibody therapeutic. As the component antibodies from which the VHHs for our candidate therapeutic are taken are camelid in origin, we propose in Specific Aim #1 to utilize?Inhibrx?s?humanization?strategy to make the antibody compatible with human administration. In Specific Aim #2 our lead cell targeting antibodies will be compared for binding in the presence of their natural substrates and to whole bacteria in order to select lead antibodies for one or both targets for development of multi-specific antibody. Finally, in Specific Aim #3, the humanized lead toxin blocking antibodies and the humanized EFB- and Protein A-binding antibodies selected in Specific Aim #2, will be combined in various formats using the Inhibrx platform. A lead therapeutic antibody will be chosen based on toxin blocking and bacterial binding capabilities. Multi-specific antibodies will then be tested in in vitro functional studies as well as in vivo (murine) models of S.a. infection. At the conclusion of Phase I, we will be ready to apply for Phase II funding to perform PK and IND enabling studies in NHPs, after which point Inhibrx will have demonstrated pre-clinical efficacy of an unparalleled anti-MRSA therapeutic.